A specific substrate from rabbit cerebellum for guanosine-3':5'-monophosphate-dependent protein kinase. III. Amino acid sequences at the two phosphorylation sites

Abstract

G-substrate is a protein present in cerebellum which is a major endogenous substrate for cyclic GMP-dependent protein kinase, and one of the few known proteins phosphorylated more effectively by cyclic GMP-dependent protein kinase than by cyclic AMP-dependent protein kinase. G-substrate has been shown to be phosphorylated on two threonine residues, and the amino acid sequences surrounding these sites, which correspond to about 30% of the primary structure, are: Leu-Asn-Val-Glu-Ser-Asp-Gln-Lys-Lys-Pro-Arg-Arg-Lys-Asp-Thr(P)-Pro-Ala-Leu-His- Ile-Pro-Pro-Phe-Ile-Ser-Gly-Val-Ile-Ser-Gln-Asn SITE 1 Leu-His-Asn-Thr-Asp-Leu-Glu-Gln-Gln-Lys-Pro-Arg-Arg-Lys-Asp-Thr(P)-Pro-Ala-Leu- His-Thr-Ser-Pro-Phe-Gln-Ser-Gly-Val-Arg SITE 2 The amino acid sequences surrounding the phosphorylated residues show 18 identities over a sequence of 26 residues, and suggest that G-substrate contains an internal gene duplication. Site-1 appears to be located 17 residues from the COOH terminus of the protein. Site 1 and site 2 are phosphorylated at similar rates by cyclic GMP-dependent protein kinase. In contrast, cyclic AMP-dependent protein kinase phosphorylates site 1 4-fold more rapidly than site 2. A decapeptide sequence surrounding the phosphothreonine residues in G-substrate shows 5 identities with that surrounding the phosphothreonine residue in protein phosphatase inhibitor 1. Inhibitor 1, a specific substrate for cyclic AMP-dependent protein kinase, also resembles G-substrate in its physical properties. The possible function of G-substrate and the molecular specificities of cyclic AMP-dependent protein kinase and cyclic GMP-dependent protein kinase are discussed in the light of these results.